This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding enzymes involved in starch biosynthesis in corn plants and seeds.
Corn starch is an important component of food, feed, and industrial products. Broadly speaking, it consists of two types of glucan polymers: relatively long chained polymers with few branches known as amylose, and shorter chained but highly branched molecules called amylopectin. Its biosynthesis depends on the complex interaction of multiple enzymes (Smith, A. et al., (1995) Plant Physiol. 107:673-677; Preiss, J., (1988) Biochemistry of Plants 14:181-253). Chief among these are ADP-glucose pyrophosphorylase, which catalyzes the formation of ADP-glucose, a series of starch synthases which use ADP glucose as a substrate for polymer formation using xcex1-1-4 linkages; and several starch branching enzymes, which modify the polymer by transferring segments of polymer to other parts of the polymer using xcex1-1-6 linkages, creating branched structures. However, based on data from other starch forming plants such as potato, and on corn mutants, it is becoming clear that other enzymes also play a role in the determination of the final structure of starch. In particular, debranching enzymes such as isoamylase and pullulanase, and disproportionating enzymes not only participate in starch degradation, but also in modification of starch structure during its biosynthesis. Different models for this action have been proposed, but all share the concept that such activities, or lack thereof, change the structure of the starch produced.
This is of applied interest because changes in starch structure, such as the relative amounts of amylose and amylopectin or the degree and length of branching of amylopectin, alter its function in cooking and industrial processes. For example, starch derived from different naturally occurring mutants of corn can be shown on the one hand to differ in structure and correspondingly to differ in functional assays such as Rapid Visco analysis, which measures changes in viscosity as starch is heated and then cooled (Walker, C. E., (1988) Cereal Foods World 33:491-494). The interplay of different enzymes to produce different structures, and in turn how different structures correlate with different functionalities, is not yet completely understood. However, it is understood that changing starch structure will result in alteration in starch function which can in turn lead to new applications or reduced processing costs (certain starch functionalities can at present only be attained through expensive chemical modification of the starch).
The role of debranching enzymes in starch biosynthesis, in particular in affecting the degree of branching, indicates that over-expression or reduction of expression of such genes in corn could be used to alter branch chain distribution of corn starch. While pullulanase genes have been described from other plants (U.S. Pat. No. 5,514.576: Nakamura. Y. et al., (1996) Plunta 199(2):209-218; Renz. A. et al., (1995) EMBL Accession No. 1076269), a pullulanase gene has yet to be described for corn.
The instant invention relates to isolated nucleic acid fragments encoding a corn pullulanase. In addition, this invention relates to nucleic acid fragments that are complementary to nucleic acid fragments encoding a corn pullulanase.
In another embodiment, the instant invention relates chimeric genes encoding a corn pullulanase or nucleic acid fragments that are complementary to nucleic acid fragments encoding a corn pullulanase, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of altered levels of corn pullulanase in a transformed host cell.
In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding a corn pullulanase, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of altered levels of corn pullulanase in the transformed host cell. The transformed host cells can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and from seeds derived from such transformed plants.
An additional embodiment of the instant invention concerns a method of altering the level of expression of corn pullulanase in a transformed host cell comprising: a) transforming a host cell with the chimeric gene encoding a corn pullulanase, operably linked to suitable regulatory sequences; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of corn pullulanase in the transformed host cell.
An addition embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or substantially all of an amino acid sequence encoding a plant pullulanase.